Metabolic Pathway Engineering

A schematic of the simplified core metabolic network

Prof. S.T. Yang

Department of Chemical and Biomolecular Engineering
The Ohio State University

Industrial fermentation products

Production
(metric tons)
Citric acid
Ethanol
Glutamate
Lactic acid
Lysine
Penicillin
Xanthan gum

1,200,000
26,000,000
1,000,000
400,000
800,000
60,000
100,000

Microorganism
A. niger
S. cerevisiae
C. glutamicum
Lactobacillus sp.
C. glutamicum
P. chrysogenum
X. campestris

Separation
method
Extraction
Distillation
Crystallization
Extraction
Crystallization
Extraction
Precipitation

Fermentation
Four Types of Commercially Important
Fermentation Products

Applications
Food
Fuel
Flavoring
Food, Plastics
Feed
Drug
Food, Oil
drilling

Microbial cells (biomass)

Microbial enzymes (cell components)
Microbial metabolites
Primary metabolites (ethanol, citric acid)
Secondary metabolites (antibiotics)

Microbial transformation (steroids)

Metabolic Engineering
substrates
oxygen

Environment

Regulation of gene expression in

the metabolic network

nutrients
temperature

Metabolome

pH

ions

Proteome
Transcriptome
Genome
DNA
mRNA
Protein
Metabolite

Regulatory mechanisms constrain network functions and produce a small range of

physiologically meaningful behaviors from all allowable network functions. Reduce
extreme pathways from 80 to 2 ~ 26.
J. Theor. Biol., 221: 309-325 (2003)

JBC 277: 2805864, 2002

Metabolic Engineering
A living cell is a complex chemical reactor in
which more than 1000 independent highly
coupled enzyme-catalyzed reactions and
selective membrane transport occur.
ME is the improvement of cellular activities by
manipulating enzymatic, regulatory and transport
functions of the cell with the use of recombinant
DNA technology (Jay Bailey, 1991)
Combined regulatory/metabolic network for central metabolism in E. coli. All of the metabolic genes
considered are shown. The genes that are regulated are indicated by the color code shown in the legend.
Genes or reactions regulated by multiple regulatory proteins or molecules are shown with multiple arrows.

Metabolic Engineering
Classical strain improvement (CSI)
Random mutagenesis to accumulate genomic
alterations and screening for the phenotypes with
desirable process characteristics
Rational metabolic engineering

Metabolic Engineering
Applications
Biocatalysis and bioprocessing (fermentation strain
improvement and metabolite overproduction)
Functional genomics, signal transduction, drug
discovery, gene therapy (biological discovery and
medical research)

The directed improvement of cellular properties

through the modification of specific biochemical
reactions or the introduction of new ones, with the
use of recombinant DNA technology

Metabolic Engineering
Bioprocessing Applications
Increase Productivity by improving cell
metabolism
Product yield
Production rate
Cell growth efficiency (energy efficiency)

Eliminate (reduce) undesirable byproducts

Eliminate (reduce) feedback inhibition
Help media design

Metabolic Engineering
Recruiting heterologous activities for
strain improvement
Completion of partial pathways - Vit. C synthesis
Hybrid metabolic networks
Construct new array of enzymatic activities to
produce new products - novel antibiotics
Perfecting strains by altering nutrient uptake and
metabolite flow - eliminating end product inhibition
Transferring of promising natural motifs enhanced oxygen transfer with cloned hemoglobin
gene

Metabolic Engineering

Metabolic Engineering in
Industrial Biotechnology

Purpose (Fermentation)
To optimize a biotechnologically important process
carried out by organisms by genetic manipulations to
affect the distribution of intracellular chemical reactions
(flux)
Some Applications
Improvement of yield and productivity amino acids
Production of novel compounds - polyketides
Extension of substrate range ethanol from xylose
Development of novel biosynthetic routes indene
Improving cell growth and fermentation kinetics

Anaerobic central
metabolic pathway
of E. coli

Some Examples

Succinic acid
production in
E. coli

Glucose
PEP
ptsG
Pyruvate

Glucose-6-P
2 NAD+

Glucose
PEP
ptsG

X
Pyruvate

Glucose-6-P
Phosphoenolpyruvate

2 NADH

poxB

Phosphoenolpyruvate
NADH

CO2

Oxaloacetate
NADH
NAD+

pyc

Pyruvate
CoA

ldhL

D()-Lactate
L(+)-Lactate

Formate

H2

Malate
Acetyl-CoA

NADH

frd

Acetyl-P
adhE

NAD+

Succinate

2 NADH
2 NAD+

Ethanol

Acetyl-CoA

Acetyl-CoA

ackA

pta

Oxaloacetate
Malate

aceB Glyoxylate

Acetyl-P
X

Citrate

aceA

iclR

Acetate

sdhAB X

aceA

aceBAK

Isocitrate

Fumarate
ackA

Acetate

pdc

CO2

pta

Fumarate

CO2
pyc

ppc

NAD+

ldhA

pfl

Pyruvate

ppc

icd

CO2

2-Ketoglutarate

Succinate
SuccinylCoA

CO2

Sorona from Corn derived

1,3-Propanediol

Ethanol production from xylose in yeasts

Glucose

Xylose
XR

NAD(P)+

Fru-6P

Glucose-6P

Ery-4P

GND1

TAL1

NAD+

XDH

CO2

XYL2

Sed-7P

Gly-3P

Ribulose-5P

XI
XylA

Xylitol
ZWF1

J. Polymers and the Environment,

Vol. 13, No. 2, April 2005

NAD(P)H

XYL1

NADH

Xylulose

TKL1

RKI1

Ribose-5P

Xylulose-5P

XYL3, XKS1

Pyruvate
TCA
cycle

Other
metabolites

Ethanol

DuPonts Sorona fermentation plant

E. coli (10-year genetic engineering work)
Reactor: Bubble column (30 m tall)
Capacity: 100,000 lbs/yr
Fermentation performance:
Volumetric productivity: 3.5 g/L/h
Product concentration: 135 g/L
Yield: 0.51 g/g glucose

1,31,3-Propanediol from Glucose in E. coli

Glucose
PEP-dependent
glucose transport

ATP-dependent
glucose transport

PEP, ATP

Pure L-(+)-Lactic Acid from

L. helveticus

2 ATP

tpi

DHAP
DAR1

GAP
NADH

gap

GPP2

Glycerol

glpK gldA

dhaB1-3

TCA cycle and

respiration
(Cell mass and NADH, etc.)

3-hydroxypropionaldehyde
NADPH
yqhD

1,3-propanediol

Replacement of the ldhD structural gene with ldhL. The overlapping

oligonucleotides used in constructing the mRNA joint between the ldhD
promoter region and the ldhL structural gene are shown. PldhD and tslpA refer
to the ldhD promoter region and slpA transcription terminator, respectively.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 66: 38353841 (2000)

Lactic acid production in yeast

Polyhydroxybutyrate (PHB)

Glucose
HMP
EMP
NAD+

Lactate

2 NAD+

Ethanol
NAD+

2 NADH

NADH

Pyruvate

ADH

NADH

Acetaldehyde

LDH

PDC

X
Plasmid pEPL2

PDH

NAD(P)+

AldDH
transport
between
cytosol and
mitochondria

Acetyl-CoA

TCA cycle

NAD(P)H

Acetate
ACS

Acetyl-CoA

Kluyveromyces lactis

Biosynthetic pathway of poly(3-hydroxybutyrate). P(3HB) is synthesized by the

successive action of b-ketoacyl-CoA thiolase (phbA), acetoacetyl-CoA reductase (phbB)
and PHB polymerase (phbC) in a three-step pathway. The genes of the phbCAB operon
encode the three enzymes. The promoter (P) upstream of phbC transcribes the complete
operon (phbCAB). Bioresource Technology 87 (2003) 137146.

Simple media
Low pH

PHA

Glucose

Alkanoates

Glycerol

Propionic acid

Acetic acid

PEP
CoA

Pyruvate

Oxaloacetate

CoA

Acetyl-CoA

TCA cycle

FadD
is
id es
ac nth
tty sy
Fa vo
Acyl-CoA
no
de
FadA
FadE

Fatty acid

3-Keto -oxidation
acyl-CoA

PhaA

Citrate

Succinyl-CoA

Indigo

Fatty acids

FadB

Acetoacetyl-CoA

Sbm

(R)-Mythyl-malonyl-CoA
YgfG

PhaB

(R)-3-Hydroxy
butyryl-CoA

Propionyl-CoA
PhaA

3-Keto-valeryl-CoA
PhaC

PhaB

3-Hydroxyl-valeryl-CoA
PhaC

P(3HB-co-3HV)

PhaB
FadG

epimerase

(R)-3-Hydroxy
acyl-CoA
PhaC

P(3HB)

PHAMCL
P(3HB-co-3HAMCL)

PP pathway

Tryptophan

Transketolase (tktA)

EMP
pathway

E4P

Tryptophanase
(tnaA)
Tryptophan
synthase
(trpA)

DAHP synthase
(aroGfbr)

Trans-2Enoyl-CoA

FadB

(S)-3-Hydroxy
acyl-CoA

NADPH

Glucose

Indole 3-glycerol
phosphate

PEP
Pyruvate kinase
(pykA, pykF)

Indole
Naphthalene
dioxygenase (NDO)

DAHP

Indoxyl
[O2]

PhaJ
YfcX

Tryptophan
synthase (trpB)

Pyruvate

Isatin

MaoC

Isatin hydrolase

TCA cycle
Indigo
Isatic acid

Indirubin

Indigo biosynthetic pathway created by the merger of indole biosynthesis and NDO
activity in one organism

-lactame Antibiotics

Glucose

NADPH

Amino Acids

Ribose-5-P

Histidine

3-PG

PP pathway

Tryptophan
Phenylalanine
Tyrosine

Erythrose-4-P

NADPH
NADP+

Phosphoenolpyruvate
PK
PEPC

Pyruvate PDH

biotin

Acetyl-CoA

DtsR

PC

Aspartate

-AAA

Citrate

ACV synthetase

pcbC IPN
synthase POA

PAA

Penicillin G
2-Oxoglutarate

HDH

NH3 NADPH

ODHC odhA

Succinyl-CoA

thrB HK

Isopenicillin N

GDH

cefD

AA

Chemical ring
expansion

Penicillin
acylase

Threonine

Ad-7-ADCA

cefEF

Ad-7-ACA

cefF

DAC

cefG

Cephalosporin C

cmcH

Acylase

Methionine
TD

Penicillin V

penDE

Penicillin N
DAOC

cefE

OCDAC
7-ACA

2,6-diaminopimelate

-AAA

cefE

Ad-6-APA

Phenylacetyl-7-ADCA

Glutamate

Homoserine-P

Pyruvate
NADP+

TCA cycle

2,3-Dihydrodipicolinate hom
Homoserine
NH3

NADPH

L-Valine

ACV

Malate

Aspartate-4-semialdehyde

NADP+

L-Cysteine
pcbAB

CS

4-Aspartylphosphate

NADPH

LAT

-AAA

P6C

Oxaloacetate

lysC AsK

dapA

Fatty acid

Lysine

lat

7-ADCA

Isoleucine

cmcI

HOCDAC

cmcJ

Cephamycin C

Lysine

Metabolic Engineering

Metabolic Engineering
Redirecting metabolite flow
Directing traffic toward the desired
branch
Reducing competition for a limiting
resource
Revising metabolic regulation

Procedures

Determine target gene (genes)

Genetic modifications
Analysis of metabolic consequences of the changes
Choice of next gene modifications

Challenges
Difficult to target the gene (or genes) and to predict
the consequences of the changes in the metabolic
pathway

Metabolic Engineering
Uncertain results due to complicated metabolic
pathways that are highly regulated by a myriad
of genes and enzymes of which many may still
not known
Success usually came from many trials after
long research and hard development efforts
costly and time consuming
It is more challenging when there is limited
knowledge on the organism and its genomics
and metabolic pathway

Metabolic Engineering
Gene targeting
Overexpression of native genes
Gene knock-out
Expression of heterologous genes

Modeling and Analysis

Metabolic flux analysis
Metabolic control analysis
Metabolic network analysis
- Flux control analysis
- Pathway analysis

The goal is to develop some principles and engineering

tools (mathematical models) that can guide the choice of
useful genetic alteration and predict its consequences

Approaches / Tools
Stoichiometric analysis of metabolic
(fermentation) pathway (mass balance)
Thermodynamic analysis of energetics of
enzyme reactions (energy balance)
Metabolic control (flux) analysis (reaction
kinetics)

Metabolic Flux Analysis

Zhang et al., Biochem. Eng. J. 2003;16:211-220

Genetic Modifications
Hypothesis

Metabolic Engineering

Mutant strains

Metabolic Characterization
Data

Metabolite profiling
- extracellular metabolites
- isotopomer intracellular metabolites
Transcriptomics - cDNA microarrays
Proteomics - 2D-gel electrophoresis

Comparison of gene expression profiles

Comparison of the expression profiles of genes for enzymes that participate in key metabolic processes
involved in the utilization of metabolites during glucose exhaustion in T. reesei and S. cerevisiae. Red
and green boxes represent those genes whose expression increases and decreases, respectively, upon glucose
exhaustion. White boxes indicate those genes that are unaffected. Yellow boxes represent genes that have yet
been not isolated from T. reesei. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 277: 1398313988, 2002.

In Silico Modeling

Proteome Profiling

Han, M.-J., S.Y. Lee. 2003. Proteome profiling and its use in metabolic and
cellular engineering. Proteomics 3: 2317-2324.

In Silico Modeling

In silico modeling of metabolism and transcriptional regulation using the constraints-based

approach. A, the constraints based approach to metabolic modeling. Flux-balance analysis can be
used to identify particular optimal solutions (such as optimization of growth) within the space (blue
point). B, transcriptional regulation reduces the steady-state solution space. (JBC 277: 2805864, 2002)

Genome-Based Modeling

Genome Shuffling

In Silico Analysis
Metabolic network reconstruction

Methodology of genome-based reconstruction of a classically derived production strain. Candidates for the relevant
mutations are introduced one by one from the relevant terminal pathways to central metabolism into the wild-type genome by
allelic replacement. Only the relevant mutations (open squares) are saved to generate a defined mutant with the minimal
mutation set that is necessary and sufficient for high-level production (minimal mutation strain)

10